Nucleofection of human embryonic stem cells
Nucleofection of human embryonic stem cells
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DOI：
10.1089/scd.2005.14.378
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发表时间：
2005-08-01
影响因子：
4
通讯作者：
Brüstle, O
中科院分区：
文献类型：
--
作者：
Siemen, H;Nix, M;Brüstle, O
人类胚胎干细胞(HES)是研究人类发育、疾病和组织再生的重要工具。为了充分利用HES细胞以用于这些应用，需要有效的基因改造技术。在这里，我们提出了一种定制的方案，用于利用Nucleofector(TM)技术转导HES细胞，并将其效率与传统的电穿孔和脂质体感染进行比较。用增强型绿色荧光蛋白(EGFP)报告载体定量检测细胞存活率和转染率。我们优化的核连接参数获得了70%的存活率。在此条件下，66%的存活细胞在核分裂后24小时有转基因表达。转基因细胞保持多能相关标记Tra-1-60、Tra-1-81和Oct4的表达，并可扩增成稳定表达转基因的克隆。核分离所需的少量HES细胞和DNA使该方法成为小型化高通量筛选(HTS)应用的一种有吸引力的工具。
Human embryonic stem ( hES) cells provide an important tool for the study of human development, disease, and tissue regeneration. Technologies for efficient genetic modification are required to exploit hES cells fully for these applications. Here we present a customized protocol for the transfection of hES cells with the Nucleofector (TM) technology and compare its efficiency with conventional electroporation and lipofection. Cell survival and transfection efficiency were quantified using an enhanced green fluorescent protein ( EGFP) reporter construct. Our optimized nucleofection parameters yielded survival rates > 70%. Under these conditions, 66% of the surviving cells showed transgene expression 24 h after nucleofection. Transfected cells maintained expression of the pluripotency associated markers Tra- 1- 60, Tra- 1- 81, and Oct4 and could be expanded to stably transgeneexpressing clones. The low quantities of hES cells and DNA required for nucleofection could make this method an attractive tool for miniaturized high throughput screening ( HTS) applications.